Pulmonary surfactant (PS) lines the alveolar epithelium of mature mammalian lungs. Natural PS has been described as a "lipoprotein complex" because it contains both phospholipids and apoproteins that interact to reduce surface tension at the lung air-liquid interface.
Since the discovery of pulmonary surfactant, and the subsequent finding that its deficiency was the primary cause of neonatal respiratory distress syndrome (RDS), a number of studies have been directed towards developing effective surfactant replacement therapy for affected individuals, particularly infants, using exogenous PS. For instance, improvements in lung function as measured by a decrease in mean airway pressure and oxygen requirements have been demonstrated using exogenous surfactants in human pre-term infants. See Hallman, et al, Pediatrics 71: 473-482 (1983); Merritt, et al, J. Pediatr. 108: 741-745 (1986); Hallman, et al, J. Pediatr. 106: 963-969 (1985); Morley, et al, Lancet 1: 64-68 (1981); Merritt, et al, New England J. Med. 315: 785-790 (1986), Smyth, et al, Pediatrics 71: 913-917 (1983); Enhorning, et al, Pediatrics 76: 145-153 (1985); Fujiwara, et al, The Lancet 1: 55-59 (1980); Kwong, et al, Pediatrics 76: 585-592 (1985); Shapiro, et al, Pediatrics 76: 593-599 (1985); Fujiwara, in "Pulmonary Surfactant", Robertson, B., Van Golde, L. M. G., Batenburg J. (eds), Elsevier Science Publishers, Amsterdam, pp. 479-503, (1984).
From a pharmacologic point of view, the optimal exogenous PS to use in the treatment of RDS would be one completely synthesized in the laboratory, under controlled and sterile conditions, with negligible batch-to-batch variability in properties. To minimize the possibility of immunologic complications, the apoprotein component of an exogenous PS should be identical to that found in humans. Unfortunately, the composition of naturally occurring PS is complex, and the art has not yet identified all of the biochemical components that generate the biophysical properties needed for high physiologic activity in lungs. In particular, the art has failed to characterize all of the apoproteins present in natural PS or identify the function of the PS apoproteins presently known.
It should be noted that the literature on PS apoproteins and their roles in surfactant function is complex, inconsistent and sometimes contradictory because heterogenous apoprotein preparations were used in many studies. To date, the art has not definitively established the number of different apoproteins present in natural PS.
Of particular interest to the present invention is the use of a low molecular weight (LMW) human PS-associated apoprotein as a component in an exogenous surfactant. Several studies have attempted to isolated or define human PS LMW apoproteins using biochemical techniques. See, for example, Phizackerley, et al, Biochem. J. 183: 731-736 (1979), Revak, et al, Am. Rev. Resp. Dis. 134: 1258-1265 (1986), Suzuki, et al, Eur. J. Respir. Dis. 69: 335-345 (1986), Taeusch, et al, Pediatrics 77: 572-581 (1986), Yu, et al, Biochem. J. 236: 85-89 (1986), Whitsett, et al, Pediatric Res. 20: 460-467 (1986), Whitsett, et al, Pediatric Res. 20: 744-749 (1986), Takahashi, et al, Biochem. Biophys. Res. Comm. 135: 527-532 (1986), Suzuki, et al, Exp. Lung. Res. 11: 61-73 (1986), Curstedt, et al, Eur. J. Biochem. 168: 255-262 (1987), Notter, et al, Chem. Phys. Lipids 44: 1-17 (1987), and Phelps, et al, Am. Rev. Respir. Dis. 135: 1112-1117 (1987).
Recently, the art has begun to apply the methods of recombinant DNA technology to overcome the problems associated with not being able to isolate to homogeneity the individual LMW PS apoproteins. For instance, Glasser, et al, Proc. Natl. Acad. Sci., U.S.A. 84: 4007-4011 (1987) reported a cDNA derived sequence of amino acid residues that forms at least a portion of a human precursor protein from which at least one mature LMW apoprotein, which they designated SPL (Phe), is formed. While Glasser, et al. were not able to determine the carboxy-terminal residue of SPL(Phe), and therefore were not able to identify its complete sequence, they did predict that mature SPL(Phe) was about 60 amino acids in length.
Jacobs, et al, J. Biol. Chem. 262: 9808-9811 (1987) have described a cDNA and derived amino acid residue sequence for a human precursor protein similar to that described by Glasser, et al, supra. However, according to Jacobs et al. the mature LMW apoprotein, which they designated PSP-B, formed from the precursor would be 76 amino acid residues in length. In addition, Jacobs, et al, noted that it was not clear that any PS apoprotein derived from the reported precursor protein was present in the surfactant preparations that had been studied clinically by others.
From the foregoing it can be seen that the literature contains multiple nomenclature for what is apparently the same PS apoprotein. Therefore, for ease of discussion, the mature apoprotein derived from the precursor protein described by Glasser, et al, supra, and Jacobs, et al, supra, will be referred to herein generically as "SP18", with the monomeric and dimeric forms being referred to as "SP18 monomer" and "SP18 dimer" respectively, when appropriate.
The canine SP18 precursor has been described by Hawgood, et al, Proc. Natl. Acad. Sci. U.S.A. 84: 66-70 (1987) and Schilling, et al, International Patent Application WO 86/03408. However, it should be noted that both those studies suffered the same inability to define the mature, biologically active form of SP18 as the Glasser, et al, supra, and Jacobs, et al, supra, studies.
Warr, et al, Proc. Natl. Acad. Sci., U.S.A. 84: 7915-7919 (1987) describe a cDNA derived sequence of 197 amino acid residues that forms a precursor protein from which a mature LMW apoprotein, they designate as SP5, is formed. Like the studies attempting to describe SP18, Warr, et al, were unable to determine the carboxy terminal residue of the mature protein formed from the precursor protein sequence, and thus were not able to definitively characterize SP5.
Because the amino acid residue sequence of the precursor protein reported by Warr et al. is different from that reported by Glasser, et al, and Jacobs, et al, it therefore appears that the art has determined that natural PS contains at least two LMW apoproteins. However, the biologically active forms of those proteins has remained undetermined.